A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts.

Department of Plant Pathology, University of California at Davis, Davis, California, USA. rbart@ucdavis.edu

Abstract

BACKGROUND:

Transient assays using protoplasts are ideal for processing large quantities of genetic data coming out of hi-throughput assays. Previously, protoplasts have routinely been prepared from dicot tissue or cell suspension cultures and yet a good system for rice protoplast isolation and manipulation is lacking.

RESULTS:

We have established a rice seedling protoplast system designed for the rapid characterization of large numbers of genes. We report optimized methods for protoplast isolation from 7-14 day old etiolated rice seedlings. We show that the reporter genes luciferase GL2 and GUS are maximally expressed approximately 20 h after polyethylene glycol (PEG)-mediated transformation into protoplasts. In addition we found that transformation efficiency varied significantly with plasmid size. Five micrograms of a 4.5 kb plasmid resulted in 60-70% transformation efficiency. In contrast, using 50 microg of a 12 kb plasmid we obtained a maximum of 25-30% efficiency. We also show that short interfering RNAs (siRNAs) can be used to silence exogenous genes quickly and efficiently. An siRNA targeting luciferase resulted in a significant level of silencing after only 3 hours and up to an 83% decrease in expression. We have also isolated protoplasts from cells prepared from fully green tissue. These green tissue-derived protoplasts can be transformed to express high levels of luciferase activity and should be useful for assaying light sensitive cellular processes.

CONCLUSION:

We report a system for isolation, transformation and gene silencing of etiolated rice leaf and stem-derived protoplasts. Additionally, we have extended the technology to protoplasts isolated from fully green tissue. The protoplast system will bridge the gap between hi-throughput assays and functional biology as it can be used to quickly study large number of genes for which the function is unknown.

Rice leaf protoplasts transformed with varying concentrations of a plasmid containing GFP. Protoplasts were prepared from 2-week-old kitaake plants and transformed as described (Methods) with p35S-GFP. Images were taken at 40× magnification under either bright field (top) or a GFP specific filter (bottom). Pictures are representative of two independent experiments for each concentration of plasmid DNA.

Luciferase and GUS expression over time. Protoplasts were prepared from 2-week-old kitaake plants and transformed as described (Methods). Luciferase (Top) or GUS (Bottom) activity was measured at 0, 4, 8, 14, 20, and 24 h post transformation. At each time point, cells were lysed by vortexing for one minute in lysis buffer (Methods) and frozen at -20°C until the experiment was complete. After the final time point, reporter gene activity was measured for all samples as described in Methods. Error bars represent 3 replicates at every time point.

Silencing of the luciferase gene by siRNAs at different concentrations. Protoplasts were prepared from 2-week-old kitaake plants and transformed as described (Methods). Varying concentrations of siRNA to luciferase (0 μg, 0.3 μg, 1 μg, 2 μg, 3 μg, 5 μg, 6 μg and 10 μg) were transformed into protoplasts along with pLUC (10 μg) and pGUS (10 μg) as an internal control. Relative luciferase activity values for each concentration of siRNA were normalized to the observed GUS expression from the same set of protoplasts. Two independent experiments (separate preparations of protoplasts) were performed with similar outcomes. Each experiment contained two replicates for each concentration of siRNAs.

siRNA silencing over time. Protoplasts were prepared from 2-week-old kitaake plants and transformed as described (Methods). Top line: 5 μg of pLUC transformed into protoplasts as described. Bottom line: 5 μg pLUC and 3 μg of siRNA to luciferase co-transformed into protoplasts. Luciferase activity was measured after 3,6, 13 and 19 h. Each time point represents 3 replicates and standard errors are displayed.

Isolation of protoplasts from green tissue. Protoplasts were prepared from 2-week-old kitaake plants and transformed as described (Methods). Picture was taken under bright field light using a Zeiss Axiovert 25 microscope with a 40× objective. Larger clear cells are derived from stem tissue whereas smaller chloroplast-filled cells were derived from green leaf tissue.

Transformation and silencing in protoplasts prepared from green tissue. Protoplasts were prepared as described (Methods). Protoplasts were suspended in Mmg solution at a concentration of 5 × 106 cells/ml. For transformation, 5 μg pLUC and 5 μg pGUS (as an internal control) were combined with 100 μl (5 × 106 cells/ml) of protoplasts and with or without 3 μg siLUC. Luciferase activity was measured as described 16 h after transformation.